Suppression of nuclear factor-kappab dependent processes using oligonucleotides

ABSTRACT

Antisense oligonucleotides which hybridize with nuclear factor- κ B(NF- κ B) mRNA and methods of using these oligonucleotides.

[0001] This application is a continuation-in-part of application Ser.No. 07/887,331, filed May 22, 1992. This invention was made withGovernment support under Grant No. CA 50234 and MH 47680, awarded by theNational Institutes of Health. The Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates generally to methods and compositionsuseful in inhibiting disorders dependent upon expression of theinducible transcription factor NF-_(κ)B.

[0004] 2. Description of Related Art

[0005] The NF-_(κ)B transcription activator is a multiprotein complexwhich can rapidly induce the synthesis of defense and signallingproteins upon exposure of cells to a wide variety of mostly pathogenicagents. Three protein subunits, I_(κ)B, p50, and p65, control thebiological functions of NF-_(κ)B. I_(κ)B is a 35-43 kDa subunit whichinhibits the DNA-binding of NF-_(κ)B and serves to retain NF-_(κ)B in aninducible form in the cytoplasm of unstimulated cells. Upon stimulationof cells, I_(κ)B dissociates from the inactive complex with p65 and p50.The released p50-p65 complex can then migrate into the nucleus andpotently transactivate genes. p50, one of the two DNA-binding subunitsof NF-_(κ)B, serves to recognize the more highly conserved half site indecameric sequence motifs with the consensus sequence GGGRNNYYCC. p50has homologies to the ret and dorsal proteins, both of which are alsoinvolved in cytoplasmic/nuclear signalling, and is identical to factorsknown as KBF1, H2TF1 and EBP-1. p50 is synthesized as an inactiveprecursor of 110 kDA.

[0006] The other DNA binding subunit, p65, binds to the less conservedhalf site of kB motifs and is apparently also homologous to rel/dorsalproteins. The combination of p50 and p65 DNA binding subunits inNF-_(κ)B extends the repertoire of binding motifs recognized with highaffinity. Only p65 appears to bind I_(κ)B. The inducibility of NF-_(κ)Bis thus equally dependent on the presence of p65 as of I_(κ)B. TheNF-_(κ)B system is so far unique among transcription factors in that theinteraction of three distinct subunits control multiple regulatorycharacteristics of a transcription activator including the subcellularlocalization and the activity, inducibility, and specificity of DNAbinding.

[0007] Many different binding sites for NF-_(κ)B have been characterizedand for most the base requirements for protein binding have been definedby methylation interference analysis or other footprinting techniques(Baeuerle, Biochem Biophys. Act., 1072:63, 1991). Most binding sites aredecameric, but some are undeca- or dodecameric. The latter may arisefrom NF-_(κ)B binding in a mutually exclusive manner to two or threedecameric motifs within the longer sequences. An alternative explanationis that p50-p65 or the p50 dimer can contact 10, 11 or even 12 basepairs. Support for the latter hypothesis comes from mutational analysisof the enhancer of the MHC class I gene H-2K^(b).

[0008] Among those disorders which are linked to activation of NF-_(κ)Bis leukemia caused by the retrovirus HTLV-1. Human T-cell leukemia virus(HTLV-1) is recognized as the etiologic agent of the human malignancy,adult T cell leukemia (B. J. Poiesz, Proc. Natl. Acad. Sci. USA,77:7415, 1980; D. J. Slamon, et al., Science, 226:61, 1984; W. C. Goh,et al., ibid. 297:1227, 1985).

[0009] Circumstantial data has implicated the HTLV-I encoded tax gene inleukemo-genesis. This gene encodes a 40kD protein that causestranscriptional transactivation of viral gene expression and alsoactivates expression of certain cellular genes that are important forgrowth (A. J. Cann, et al., Nature 318:571, 1985; B. K. Felber, et al.,Science, 229:675, 1985; J. Fujisawa, et al., Embo Journal 5:713, 1986).In vitro studies have demonstrated that tax can activate the promotersof the interleukin 2 receptor (IL-2R) a-chain, GM-CSF, fos, PDGF, IL-6,NGF, TGF-_(β), HIV LTR as well as its own LTR (D. J. Slamon et al.,Science, 226:61, 1984; W. C. Goh et al., ibid., 227:1227, 1985; J.Inoue, et al., Embo Journal 5:2883, 1986; S. Miyatake, et al., Mol.Cell. Biol. 8:5581, 1988; K. Nagata, et al., J. Virol. 63:3220, 1989; L.Ratner, Nucleic Acid Research, 17:4101, 1989; J. Sodroski, et al.,Science, 228:1430, 1985; J.E. Green, et al., Mol. Cell. Biol. 11:4635,1991; S. J. Kim, et al., J. Exp. Med., 172:121, 1990; E. Bohnlein, etal., J. Virol., 63, 1578, 1988). In vitro studies of tax effects on geneexpression, have demonstrated two independent pathways for its action ontranscription (M. R. Smith and W. C. Green, J. Clin. Invest. 87:761,1991). The first affects the family of nuclear transcription factorsrelated to c-rel which bind to NF-_(κ)B sites and are important for thenormal activation of lymphocytes. NF-_(κ)B response sequences occur in anumber of genes including the HIV LTR (G. Nable, et al., Nature London,326:711, 1987) and the IL-6 promoter (T. A. Libermann, et al., Mol.Cell. Biol., 10:2327, 1990). The heterodimer composed of the p5C and p65rel related proteins have been shown to affect the transcription of manyof these genes (P. A. Baeuerle, Biochem. Biophys. Acta, 1072:63, 1991).The other effect of tax is thought to be NF-_(κ)B independent, wherebytax activates its own promoter through three tax responsive elements(TREs). Similar sequence motifs have been identified in fos (M. Fujii etal., Proc. Natl. Acad. Sci., USA, 85:8526, 1988), an immediate earlyresponse gene.

[0010] Unfortunately, although it is known that tax and other disordersappear linked to activation of NF-_(κ)B, no therapeutic modalities existwhich can suppress this activation and thereby inhibit progression orestablishment of the disorder. The present invention addresses this needand provides composition and means of accomplishing this goal.

SUMMARY OF THE INVENTION

[0011] The present invention arose from the discovery that an antisenseoligonucleotide which hybridizes to nuclear factor-_(κ)B (NF-_(κ)B) mRNAcan be used to suppress processes which depend upon activation ofNF-_(κ)B.

[0012] As a consequence of this discovery, the present inventionrepresents a major improvement over existing techniques for suppressingNF-_(κ)B dependent processes which often entail use of chemicals whichare highly toxic, especially in terms of their utilization in vivo. Theantisense oligonucleotides of the invention display a high degree ofbiocompatibility with host systems such that the low efficiency andtoxic aspects of prior art methodologies and compositions is avoided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1A, B is a Northern analysis of early genes in tax orNF-_(κ)B antisense treated cells.

[0014]FIG. 2 is a CAT assay in the presence of NF-_(κ)B p65 antisenseODNs.

[0015]FIG. 3 is an electrophoretic mobile shift assay (EMSA) of nuclearextracts obtained from unmanipulated, sense and antisense treated cells.

[0016]FIG. 4 is growth curves of cells treated with tax or NF-_(κ)Bantisense, in vitro. Symbols are mock (No; Δ), sense (SEN; ◯) andantisense (ANT; •)

[0017]FIG. 5 shows in vivo growth inhibition of B cell line tumors byNF-_(κ)B antisense treatment.

[0018]FIG. 6 shows the effect of antisense NF-_(κ)B antisense andN-acetyl cysteine (NAC) on mouse survival after LPS challenge.

[0019]FIG. 7 shows serum IL-6 levels after LPS challenge. Error barsrepresent one standard deviation.

[0020]FIGS. 8a and 8 b show a Northern blot analysis of NF-_(κ)Bdependent gene expression in tissues of LPS challenged mice. Lanes 1-9represent pretreatment for 3 hours with NAC or antisense NF-_(κ)B. Lanes10-18 show the effects of 24 hours pretreatment.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0021] The present invention relates to an antisense oligonucleotidesequence which can hybridize to nuclear factor _(κ)B (NF-_(κ)B) subunitmRNA. This antisense sequence is highly useful for suppressing in vitroor in vivo NF-_(κ)B dependent or associated processes in individuals.Such processes are typically associated with such disorders as thosemediated by immune or cytokine responses (for example, septic shock) aswell as those disorders induced by infectious agents such asretroviruses, more specifically, HIV and HTLV.

[0022] The antisense oligonucleotides of the invention are preferablydirected to the p65 or p50 subunits NF-_(κ)B mRNAs. Most preferably, theantisense oligonucleotides are complementary to the translationinitiation nucleic acid sequence of these subunits. In general, theantisense oligonucleotides of the invention are capable of hybridizingto DNA which has the nucleotide sequence 3′-TAGCAGACGGTACCACTTCTA-5′ or3′-CTTGTCAAGCAGGTACCGGC-5′ or 3′-TTTGTCTAGCAGGTACCAGT-5′. Most preferredare antisense oligonucleotides having the nucleotide sequence5′-ATCGTCTGCCATGGTCAAGAT-3′, 5′-TCGTCTGCCATGGTCAAGAT-3′, or5′-GAACAGTTCGTCCATGGCCG-3′ or 5′-AAACAGATCGTCCATGGTCA-3′.

[0023] As a general matter, the oligonucleotide employed will have asequence that is complementary to the sequence of the target RNA.However, absolute complementarity is not required; in general, anyoligonucleotide having sufficient complementarity to form a stableduplex with the target RNA so that translation of the RNA is inhibited,is considered to be suitable. Since stable duplex formation depends onthe sequence and length of the hybridizing oligonucleotide and thedegree of complementarity between the antisense oligonucleotide and thetarget sequence, the system can tolerate less fidelity (complementarity)when longer oligonucleotides are used. However, it is presently believedthat oligonucleotides of about 8 to 40 bases in length and havingsufficient complementarity to form a duplex having a melting temperatureof greater than about 40° C. under physiologic conditions areparticularly well suited for practice of the invention (Thoung, et al.,PNAS USA, 84:5129, 1987; Wilson, et al., Nucleic Acids Res., 16:5137,1988). Accordingly, such oligonucleotides are preferred.

[0024] Another variable that may affect practice of the invention is theregion of the target RNA to which the selected oligonucleotide isdesigned to hybridize. Although oligonucleotides capable of stablyhybridizing with any region of the RNA may be suitable for practice ofthe invention, oligonucleotides complementary to a region including thetranslation initiation nucleic acid sequence of the NF-_(κ)B subunit areparticularly effective. The antisense oligonucleotide is consideredeffective as long as the translation of the mRNA to which theoligonucleotide is complementary is inhibited.

[0025] The oligonucleotide employed may be unmodified or modified.Suitable modifications include, but are not limited to, the ethyl ormethyl phosphonate modifications disclosed in U.S. Pat. No. 4,469,863and the phosphorothioate modifications deoxynucleotides described byLaPlanche, et al. (Nucleic Acids Research 14:9081, 1986), and by Stec.et al., (J. Am. Chem. Soc. 106:6077, 1984). The modification to theantisense oligonucleotide is preferably a terminal modification in the5′ or 3′ region. Preferred are modifications of the 3′ terminal regionas described herein. Furthermore, recent advances in the production ofoligoribonucleotide analogues mean that other agents may also be usedfor the purposes described here, for example, 2′-methylribonucleotides(Inoue, et al., Nucleic Acids Res. 15:6131, 1987) and chimericoligonucleotides that are composite RNA-DNA analogues (Inoue, et al.,FEBS Lett., 215:327, 1987).

[0026] Of course, in order for the cell targets to be effectivelyinhibited by the selected antisense oligonucleotides, the cells must beexposed to the oligonucleotides under conditions that facilitate theiruptake by the cells. For in vitro therapy this may be accomplished by anumber of procedures, including, for example, simple incubation of thecells with the oligonucleotides in a suitable nutrient medium for aperiod of time suitable to achieve selective inhibition of the cells.For example, where the cell targets of the antisense oligonucleotide ofthe invention are present in bone marrow cells, procedures can beemployed such as those described by Gartner and Kaplan, Proc. Natl.Acad. Sci. USA, 77:4756, 1980; Coulombel, et al., Blood 67:842, 1986;Meagher, et al., Blood, 72:273, 1988; or U.S. Pat. No. 4,721,096 with anoptimal concentration of the selected antisense oligonucleotide. Afterthe marrow cells have been exposed to the oligonucleotide and, in somecases, cultured as described above, they are then infused into thetransplant recipient to restore hemopoiesis.

[0027] The antisense oligonucleotide of the invention can also beadministered to provide in vivo therapy to a patient having a disorderwhich is associated with activation of NF-_(κ)B. Such therapy can beaccomplished by administering, in vitro and in vivo as the case may be,a therapeutically effective amount of antisense oligonucleotide, or asfurther described below, the antisense oligonucleotide in combinationwith glutathione precursor. The term “therapeutically effective” meansthat the amount of antisense oligonucleotide administered alone, or incombination with glutathione precursor, is of sufficient quantity tosuppress to some beneficial degree activation of NF-_(κ)B and thedisorder associated with activation of NF-_(κ)B. Examples of suchdisorders include those associated with LPS-induced septic shock andthose associated with elevated IL-6 production. The NF-_(κ)B process mayalso be tissue specific. For example, the disorder may be detected inthe spleen, liver, kidney, or lung. Preparations utilizing antisenseoligonucleotide can comprise the oligonucleotide in a simple buffersolution or, alternatively, in a more complex vehicle as describedbelow.

[0028] The invention also provides a method of monitoring theeffectiveness of suppressing NF-_(κ)B and NF-_(κ)B dependent processesin the tissue of an individual after administering a therapeuticallyeffective amount of NF-_(κ)B antisense comprising detecting the level ofcytokine production in a tissue, before and after antisense therapy.Preferably, the cytokine is IL-6. IL-6 can be detected by immunologicalmethods such as ELISA, or nucleic acid methods, such as Northern blotanalysis of IL-6 mRNA.

[0029] Studies have suggested that glutathione precursors may be usefulin regulating activation of NF-_(κ)B (Staal, et al., Proc. Natl. Acad.Sci. USA, 87:9943, 1990; Bruno, et al., Biochemical Pharmacology,37:4319, 1988). Typically, these precursors are an acylcysteine, such asN-acetylcysteine. These compounds appear to block activation of NF-_(κ)Bin both resting as well as activated cells. Unfortunately, thesecompounds have been found to be extremely toxic in vivo whenadministered alone. An advantage of the present system is that it allowsa means of reducing the concentration of glutathione precursors which isutilized by combining the precursor with the antisense oligonucleotideof the invention. Thus, according to the method of the invention, it ispossible to administer the antisense oligonucleotide in combination witha precursor of glutathione. The term “in combination” means that theantisense oligonucleotide and the glutathione precursor are administered(1) separately at the same or different frequency using the same ordifferent administration or (2) together in a pharmaceuticallyacceptable composition. If desired, the antisense oligonucleotide andglutathione precursor can be administered substantiallycontemporaneously. The term “substantially contemporaneously” means thatthe antisense oligonucleotide and glutathione precursor are administeredreasonably close together with respect to time, for example,simultaneously to within a few hours.

[0030] “Pharmaceutical combination” includes intimate mixtures of thetwo components of the invention, as in classical compositions, and alsonon-mixed associations, such as those found in kits or pharmaceuticalpacks.

[0031] Antisense oligonucleotide, alone or in combination withglutathione precursor, can be administered in a single dose or can beadministered in multiple doses over a period of time, generally byinjection. Various administration patterns will be apparent to thoseskilled in the art. The dosage ranges for the administration of theantisense obigonucleotide of the invention are those large enough toproduce the desired effect of suppressing the undesired NF-_(κ)Bdependent process. The dosage should not be so large as to cause adverseside effects, such as unwanted cross-reactions, anaphylactic reactions,and the like. Generally, the dosage will vary with the age, condition,sex and extent of the disease in the patient and can be determined byone of skill in the art without undue experimentation. The dosage can beadjusted by the individual physician in the event of any counterindications, immune tolerance, or similar conditions. Those of skill inthe art can readily evaluate such factors and, based on thisinformation, determine the particular therapeutically effectiveconcentration of antisense oligonucleotide, or antisense oligonucleotidein combination with glutathione precursor, to be used. Generally, dosagefor antisense oligonucleotide can vary from about 1.0 mg/g body weightto about 100 mg/g body weight, preferably from about 10 mg/g body weightto about 80 mg/g body weight, most preferably from about 30 mg/g toabout 50 mg/g body weight. Glutathione precursor, such asN-acetylcysteine, can be administered from about 1 mM to about 50 mM,more preferably from about 10 mM to about 40 mM, most preferably fromabout 20 mM to about 30 mM.

[0032] Antisense oligonucleotide and glutathione precursor can beadministered as the compound or as a pharmaceutically acceptable salt ofthe compound, alone, in combination, or in combination withpharmaceutically acceptable carriers, diluents, and vehicles. Mostpreferably, antisense oligonucleotide and glutathione precursor aremixed individually or in combination with pharmaceutically acceptablecarriers to form compositions which allow for easy dosage preparation.

[0033] The antisense oligonucleotide and glutathione precursorcomposition of the present invention can be administered in anyacceptable manner including by injection, using an implant, and; thelike. Injections and implants are preferred because they permit precisecontrol of the timing and dosage levels used for administration, withinjections being most preferred. Antisense oligonucleotide andglutathione precursor compositions according to the present inventionare preferably administered parenterally.

[0034] Antisense oligonucleotide according to the present invention canbe administered to the patient in any acceptable manner includingorally, by injection, using an implant, nasally and the like. Oraladministration includes administering the composition of the presentinvention in tablets, suspensions, implants, solutions, emulsions,capsules, powders, syrups, water compositions, and the like. Nasaladministration includes administering the composition of the presentinvention in sprays, solution, and the like. Injections and implants arepreferred because they permit precise control of the timing and dosagelevels used for administration, with injections being most preferred.Antisense oligonucleotide is preferably administered parenterally.

[0035] Glutathione precursor according to the present invention can beadministered to the animal in any acceptable manner including byinjection, using an implant, and the like. Injections and implants arepreferred because they permit precise control of the timing and dosagelevels used for administration, with injections being most preferred.Glutathione precursor is preferably administered parenterally.

[0036] Antisense oligonucleotide and glutathione precursor compositionscan be administered in an injectable formulation containing anyglutathione precursor and antisense oligonucleotide compatible andbiocompatible carrier such as various vehicles, adjuvants, additives,and diluents to achieve a composition usable as a dosage form.

[0037] Aqueous vehicles such as water having no nonvolatile pyrogens,sterile water, and bacteriostatic water are also suitable to forminjectable antisense oligonucleotides and glutathione precursorformulations. In addition to these forms of water, several other aqueousvehicles can be used. These include isotonic injection compositions thatcan be sterilized such as phosphate buffered saline, sodium chloride,Ringer's, dextrose, dextrose and sodium chloride, gelatin and lactatedRinger's. Addition of water-miscible solvents, such as methanol,ethanol, or propylene glycol generally increases solubility andstability of the compounds in these vehicles.

[0038] Nonaqueous vehicles such as cottonseed oil, squalene, sesame oil,or peanut oil and esters such as isopropyl myristate may also be used assolvent systems for glutathione precursor and antisenseoligonucleotides. Additionally various additives which enhance thestability, sterility, and isotonicity of the composition includingantimicrobial preservatives, antioxidants, chelating agents, gelatin andbuffers can be added. Any vehicle, diluent, or additive used would,however, have to be compatible with the compounds of the presentinvention.

[0039] Antisense oligonucleotides and glutathione precursor compositionsaccording to the present invention can be administered in the form of aslow-release subcutaneous implant which is inserted beneath the skin.The implant can take the form of a pellet which slowly dissolves afterbeing implanted or a biocompatible and glutathione precursor andantisense oligonucleotide compatible delivery module well known to thoseskilled in the art. Such well known dosage forms are designed such thatthe active ingredients are slowly released over a period of several daysto several weeks.

[0040] The following examples are intended to illustrate but not limitthe invention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively beadopted without resort to undue experimentation.

EXAMPLE 1 ANTISENSE INHIBITION OF TUMOR CELLS IN VITRO A. PREPARATION OFOLIGONUCLEOTIDES

[0041] Sense and antisense oligodeoxynucleotides (ODNs) were synthesizedby the phosphoramide method on an ABI automated synthesizer (FosterCity, Calif.). The phosphothioate (PS) sulfurization modification(Stein, et al., Nucleic Acids Res., 16:3209, 1988) was performed duringsynthesis, where TETD and acetonitrile were substituted for the usualiodine, pyridine and water during the oxidation step. ODNs were purifiedaccording to published procedures (Miller, et al., J. Biol Chem.,255:9659, 1980).

[0042] One set of sequences was selected to be complementary to thetranscript encoded by the HTLV-I LTR-tax cassette which had previouslybeen used to generate transgenic mice (Nerenberg, et al., Science,237:1324, 1987). The antisense sequence was GAAGTGGGCCATGTGGA*A*G(location 718-737 on HTLV-I LTR-tax construct) which included the AUGinitiation codon (underlined) and sense sequence wasCTTCCACATGGCCCACT*T*C*, the exact complement of the tax-antisense ODNabove. Asterisks show sites of PS modification.

[0043] An upstream sense primer (AAGCGTGGAGACAGTTCAGG, location 423-442)was synthesized as a primer pair for the modified or unmodifiedantisense primer (location 737-718). Similarly, a downstream antisenseoligonucleotide (TTGGCGGGGTAAGGACCTTG, location 986-1004) wassynthesized to pair with the modified or unmodified sense (location718-737).

[0044] A second set of ODNs was prepared based on the nucleotidesequences of the p50 and p65 subunits of NF-_(κ)B. The ODNs for p50NF-_(κ)B were antisense5′-ATCGTCTGCCATGGTGAAGAT-3′(human),antisense5′-TCGTCTGCCATGGTGAAGAT-3′(mouse) and sense 5′-ATCTTCACCATGGCAGCAGA-3′(human and mouse). Thecorresponding ODNs for p65 were antisense 5′-GAACAGTTCGTCCATGGCCG-3′(human), antisense 5′-AAACAGATCGTCCATGGTCA-3′ (mouse) and sense 5-°CGGCCATGGACGAACTGTTC-3′ (human) and sense5′-TGACCATGGACGATCTGTTT-3′(mouse). Initiation codons are underlined.Both sets of ODNs extended over the translational initiation sites oftax or NF-_(κ)B mRNAs.

B. ESTABLISHMENT OF TAX EXPRESSING TUMOR CELL LINES

[0045] LTR-tax transgenic mice were originally generated from CD1females and (C57BL/6×DBA/2) F1 males, and thus were genetically highlyheterogeneous (Nerenberg, et al., Science, 237:1324, 1987). One line(6-2) containing 40 copies of the transgene and expressing high levelsof tax was back bred to C57BL/6 mice for greater than 30 generations.Spontaneously arising fibroblastic tumors were sterily harvested, finelyminced and grown in bulk culture for 30 passages. Single cell cloneswere obtained by dilution and growth in Terasaki plates in the presenceof filtered conditioned medium from the bulk cultures. Individual cloneswere grown in bulk culture and tested for ability to form tumors insyngeneic animals. One of these lines (designated B line) was selectedfor further study as it rapidly and reproducibly caused tumors wheninnoculated into the hind limb of animals when 1×10⁶−1×10⁷ cells wereinjected. Tumors grew rapidly at the site of local injection and nevercaused metastasis. These tumors attracted high numbers of granulocytes,similar to the original tumors (Nerenberg, et al., Science, 237:1324,1987), presumably caused by high secretion of cytokines. Mice typicallydie from localized effects of the tumor by 2 months after injection.Other cell lines had similar gene expression phenotypes, but grew moreslowly, or required higher numbers of inoculated cells to form tumors.Cells were H-2^(B) haplotype, and expressed high levels of tax.

C. EFFECT OF OLIGONUCLEOTIDES ON TUMOR CELLS IN VITRO

[0046] Cells of the B line were cultured in T-75 flasks to 70%confluence. Cells were grown in modified DMEM (Celigro, Mediatech,Wash., D.C.) plus 10% heat inactivated fetal calf serum (FCS) (GIBCO,Grand Island, N.Y.) except where indicated. The unmodified or PSmodified ODNs were added to a final concentration of 20 mg/cc for 48hrs. The cells were pre-incubated for 6 hrs in the presence of theseODNs with 10% FCS or without serum prior to transfections. Poly A+ RNAwas extracted from tissues and digested with proteinase K and purifiedby the FastTrack method (Bradley, et al., BioTechniques, 6:114, 1988)(Invitrogen, San Diego, Calif.). Samples were denatured and run onformaldehyde 1.4% agarose gels followed by blotting onto nylon membraneand UV fixation. Cloned cDNAs (early genes) were used for generation ofprobes. Probes were labeled via the random primer method with ³²P dATPplus dCTP. Specific activities were 1×10⁹ cpm/mg. The effect ofantisense on tax or NF-_(κ)B gene expression in vitro is shown byNorthern analysis in FIG. 1, A and B, respectively.

[0047] Tax specific sense ODNs used as a control, had no effect on cellgrowth or gene expression (FIG. 1A lane 2). Previous analyses of taxantisense ODN treated cells demonstrated approximately 10 fold decreasein tax protein production, which led to a corresponding 10 fold decreasein transactivation of the HTLV-I LTR in transient assays. A specificdecrease in the levels of specific RNA can be seen, and this isconsistent with direct (CRE/ATF) activation of this promoter by tax(Fujii, et al., Proc. Natl. Acad. Sci., 85:8526, 1988). However, noalteration in the levels of many other growth related genes was seen(FIG. 1A lane 3), suggesting that they were not directly activated bytax.

[0048] In contrast, inhibition of either the p50 or p65 subunits ofNF-kB caused profound effects on endogenous cytokine gene expression(FIG. 1B). IL-6 was affected more than GM-CSF and there was no apparenteffect on c-fos, tax, or actin expression. Inhibition of p65 wasslightly more effective than p50. This result demonstrated specificityof the ODNs for the NF-kB responsive genes. To quantitate functionaleffects of this inhibition, transient CAT expression assays wereperformed on an NF-_(κ)B dependent promoter. The plasmid HIV-CAT wasused, as this retroviral LTR contains 2 copies of an NF-_(κ)B targetsequence and has shown to be highly NF-kB responsive (Nabel, et al.,Nature, 326:711, 1987).

[0049]FIG. 2 shows greater than 20 fold inhibition of CAT expression inthe presence of NF-kB p65 antisense ODNs. In these studies, cells (cellline B) were pretreated with p65 NK-kB oligonucleotides for 8 hours. Thecells were then transfected with 5 mg of an HIV LTR-CAT construct. CATactivity was analyzed after an additional 48 hours. As an internalcontrol, 5 mg of CMB-β gal (Clontech, San Francisco, Calif.) wasco-transfected and analyzed by MUG assay (MacGregor, et al., Methods inMolecular Biol., 1989). Transfections used Lipofectin (Bethesda ResearchLaboratories, Bethesda, Md.) as per Flegner, et al., Proc. Nat. Acad.Sci., 85:7413, 1989). The level of CAT activity was determined by thinlayer chromatography using ¹⁴C chloramphenicol. Results were quantitatedby scintillation counting of the extracted radiographic spots and areshown.

[0050] The findings above were further confirmed by direct assay ofprotein binding to NF-kB target sites. Electrophoretic mobility shiftanalysis (EMSA) of nuclear extracts obtained from unmanipulated, senseand antisense treated cells are shown in FIG. 3.

[0051] The target sequence for EMSA consisted of a double stranded35-mer: 5′AGCTTCAACAGAGGGGACTTTCCGAGAGGCTCGAG3′ (ODN A). The underlinedsequence is identical to the NF-_(κ)B consensus in mouse Ig k lightchain and to that used by N. Kabrun, et al., Proc. Natl. Acad. Sci.,88:1783, 1991. The second strand was synthesized by hybridization withan 11 -mer (ODN B), (complementary to the sequence not underlined) whichwas labeled at its 3′ end by klenow mediated extension in the presenceof α-³²P dATP. This yielded a double stranded labeled 35-mer. Thesequence for the mutant NF-_(κ)B was generated from a 35-mer (ODN c)sequence: 5′AGCTTCAACGAGGcGACTTTCCGAGAGGCTCGAG3′. The NF-_(κ)B site isunderlined with the mutation in small letters. This ODN was hybridizedwith ODN B and filled using klenow in the presence of unlabeled dNTPs.

[0052] For these experiments, 3 mg of nuclear extract from untreated(no) sense treated (se), or antisense treated (an) cells (cell line B)were incubated with a double stranded ODN encoding the NF-_(κ)Bconsensus target sequence (Kabrun, et al., Proc. Natl. Acad. Sci., 88:1783, 1991). 1×10⁴cpm (approx. 0.86 ng) of labeled ODN was used.Procedures were as published (Kabrun, et al., Proc. Natl. Acad. Sci.,88: 1783, 1991). Three NF-_(κ)B specific bands (I, II, III) (Baldwin, etal. Mol. Cell. Biol., 11:4943, 1991) were readily identified.Competition assays are shown in the left panel. The ratios of molarexcess of unlabeled consensus ODN (NF-_(κ)B) or mutant NF-_(κ)B(M) areshown.

[0053] Analysis of unmanipulated cells (lanes 1,6) revealed 3 bands ofNF-_(κ)B complexes similar to those previously described (A. Baldwin, etal., Mol. Cell. Biol., 11:4943,1991). Unlabeled NF-_(κ)B consensusoligonucleotide competitively inhibited all complexes (lanes 2,3). Incontrast, an oligonucleotide with a single G to C substitution in thebinding site failed to block the formation of complexes (lanes 4,5).These data confirm the specificity of this EMSA assay for detection ofNF-_(κ)B complexes. Treatment of these cells with p65 sense ODNs had noeffect on complex formation (lane 7). In contrast, use of p65 antisenseODNs specifically ablated all complex formation (lane 8). This occurredin a dose dependent manner and was also obtained with p50 specific ODNs(data not shown).

[0054] The effects of tax or NF-_(κ)B antisense ODN inhibition on invitro growth of mouse cell lines are shown in FIG. 4A. Balb/3T3 cells,or cell line B were cultured in 10 cm dishes, in the presence of varyingconcentrations of FCS (as indicated on the abscissa). 1×10⁴ cells/mlwere plated, an amount calculated to give approximately 30% confluence.20 mg/ml p65 sense or antisense ODNs were added and cultures wereallowed to grow for 6 days. ODNs were replenished at 3 day intervals.Cells were counted and expressed as % confluence. Mock treatment (No;Δ), sense (SEN; ◯) and antisense (ANT; •) are indicated. For each cellline, mock, sense and antisense PS ODN treatments were performed. Aconstant amount of ODN (20 mg/ml) was used, and growth rates weremeasured daily. The data in FIG. 4 shows the percent confluence at day 6as a function of varying serum concentration. Serum concentration hasprofound effects on the growth rate of cells and previous studies havedemonstrated induction of nuclear translocation of NF-_(κ)B by serum (T.A. Libermann, et al., Mol. Cell. Biol., 10:2327, 1990). Therefore, thisallowed the effects of ODNs to be displayed over a wide range of growthconditions. Treatment with tax specific ODNs, which caused a 90%inhibition of tax expression, had no apparent effects on growth rates ofcells. In contrast, p65 antisense ODNs had profound effects on thegrowth of cell line B, with no apparent effect on Balb/3T3 growth at allserum concentrations. Similar results were obtained when theconcentration of NF-_(κ)B ODN was varied and serum concentration wasfixed, or with other tax expressing fibrosarcoma lines. p50 ODNs wereslightly less effective at the same concentration but gave a similarprofile. Mixtures of both p50 and p65 ODNs had an additive inhibitoryeffect. It is likely that additional HTLV-I encoded proteins other thantax are necessary for transformation by the native virus. Thus, tax andNF-_(κ)B may play different roles in maintenance of transformation ofhuman lymphocytes than that demonstrated for mouse fibroblasts. Todetermine the effects of tax and NF-_(κ)B antisense ODNs on lymphocytes,the HTLV-I transformed human cell line, MT2 (M. Kozac, Nature, 308:241,1984) was analyzed. FIG. 2B shows a profile of growth inhibition similarto that seen for the HTLV-I transformed murine cell line MT-2 (Kozac,Nature, 308:241, 1984). Cells were grown in 4% FCS in the presence orabsence of NF-_(κ)B or tax ODNs. Synthesis of new PS ODN was required,as the 5′ end of the tax translation initiation target sequence in thetransgenic mice varied slightly from that in the native human virus.(The sequence for inhibition of tax in human cells is antisense5′-TCGTCTGCCATGGTGAAGAT-3′.) Cells were allowed to grow for up to 20days. Growth is expressed as absolute cell number in triplicate 6 welldishes. Tax antisense ODNs had no apparent effect on growth of theinfected human cell line (left panel), despite significant inhibition oftax protein. In contrast, p65 antisense ODNs profoundly inhibited growthat all time points.

EXAMPLE 2 ANTISENSE INHIBITION OF TUMOR GROWTH IN VIVO

[0055] Studies were performed on the effects of ODN mediated suppressionof unmanipulated line B cells which were transferred to syngeneicC57B1/6 mice. Once tumors were established (7 days after injection inthe hind limb), mice were treated with 3 intraperitoneal (IP) injectionsat 3 day intervals of 40 mg/g of sense and antisense ODNs as describedby Goodchild, et al. (T. A. Libermann, et al., Mol. Cell. Biol.,10:2327, 1990). Western blot analysis of tumors exposed to tax antisenseODN treatment showed virtually complete suppression of tax expression atthe 40 mg/g dose. As previously seen above in in vitro studies, noperturbation in growth rate of tumors was seen in the animals treatedwith tax antisense ODNs. Treatment with antisense ODN to NF-_(κ)B haddramatically different effects. FIG. 5 shows a typical comparison oftypical matched mice either 15 days after treatment with sense (left) orantisense (right) ODNs to p65. In each case, treatment with antisense top65 caused regression of tumors, while treatment with sense caused nodiminution in growth. A total of eight antisense treated and eightcontrols were analyzed over time, and growth rates were determined byweighing excised tumors. The results are shown in Table 1. TABLE 1 TIMECOURSE ANALYSIS OF NF-_(κ)B p65 SENSE AND ANTISENSE TREATED MICE DAYSAFTER START TREATMENT OF TREATMENT NONE SENSE ANTISENSE 0  348 ± 214^(a)N.D.^(b) N.D. 4 799 ± 343 748 ± 424 260 ± 198 8 1385 ± 774  1264 ± 671 122 ± 84  15 5394 ± 2864 4874 ± 2571 45 ± 28 60 N.D. 7243 ± 3872 <45^(c)

[0056] Clear growth inhibition was seen as early as 8 days after firsttreatment with profound differences occurring by 15 days. Histologicanalysis of these tumors was also performed. Samples obtained from micetreated with sense NF-_(κ)B ODN revealed typical morphologycharacteristic of growing tumors. Treatment with antisense ODN showedfocal tumor necrosis with inflammatory infiltrate by 4 days, followed bywidespread segmental necrosis by 7 days. By 10 days, it fibrotic tissueand tumor capsule with occasional inflammatory cells were aii thatremained. Beyond 10 days, it was difficult to identify the tumor site.Untreated mice, or those treated with sense ODNs, died between 8 and 12weeks, while antisense ODN treated mice have been followed for up to 5months without evidence of recurrence of tumors. In none of these micewere ODN injections given beyond the first 9 days.

[0057] The present studies reveal striking similarities between theeffects of tax on mouse fibroblasts and HTLV-I or II virus on humanT-cells. Though tax is necessary for transformation of mouse fibroblastsor human T-cells, the present studies indicate that continued highlevels of tax expression (greater than 10% of the unmanipulated tumor)is not necessary for maintenance of the activated phenotype, or forgrowth of these cells. Similarly, HTLV-I associated human lymphomasfrequently express very low levels of tax (T. Kinoshita, et al., Proc.Natl. Acad. Sci. USA., 86:5620, 1989). In contrast, tumor growth is verysensitive to the levels of NF-_(κ)B expression. It appears that fullytransformed mouse or human cells take up sufficient ODNs to effect tumorregression at doses of ODN which are well tolerated in vivo. In fact,previous toxicity studies suggest that mice readily tolerate at least2.5 times amounts used here (J. Goodchild, et al., Proc. Natl. Acad.Sci. USA., 85:5507, 1988). The basis for this difference insusceptibility of tax transformed and normal cells to NF-_(κ)B ODNs isunclear. It may represent a true difference in the requirement ofintracellular NF-_(κ)B for survival. Alternatively, it may reflect adifference in the ability to internalize the ODNs. Previous studies havecorrelated the activation/growth state of cells with their ability totake up oligonucleotides (R. M. Crooke, Anti-Cancer Drug Design, 6:609,1991; P. Iversen, ibid 6.531, 1991), and this process appears to occurvia receptor mediated endocytosis. Tax or virus transformed tumor cellsappear highly activated, which may cause them to take up more ODNs.Preliminary data supports this possibility.

[0058] Histologic analysis reported here showed profound and widespreadtumor necrosis, and even this short window of treatment was apparentlysufficient to prevent tumor recurrences. This suggests that these ODNsmay provide a valuable approach to therapy of HTLV-I associated adultT-cell leukemia which has proved largely refractory to other modalities.In addition, these antisense sequences should be able to inhibit otherprocesses which rely upon activation of NF-_(κ)B.

EXAMPLE 3 ANTISENSE NF-_(κ)B ODNs PREVENT LPS MEDIATED DEATH

[0059] In order to compare the effects of NAC or NF-_(κ)B AS-ODNs on thetoxic manifestations of LPS induced septic shock, survival rates for thedifferent treatment groups of mice were compared. This data issummarized in FIG. 6.

[0060] LPS-induced lethality. In vivo LPS challenge was performedaccording to the modified methods of Broner et al., Critical CareMedicine 16:848, 1988; and Peristeris, et al., Cellular Immunology,140:390, 1992. A total of 35 C57BI/6 mice (body weight 25-35g) werestudied in three treatment groups: 1) 10 mice were treated with LPSfollowed by two mock injections of saline. 2) 10 mice were pretreatedwith NAC 12 hours and ½ hour prior to innoculation with LPS. 3) 10 micewere pretreated with NF-_(κ)B antisense ODNs 20 hours and ½ hour priorto LPS, and 5 mice were treated 20 and ½ hours prior to LPS with anirrelevant HTLV-I specific antisense ODN (Kitajima, I., et al., J. Biol.Chem. 267: 25881, 1992) as a control.

[0061] LPS (Escherichia coli 055:B5, Sigma, St. Louis., Mo.) was givenas a single dose by intraperitoneal (ip) injection. Optimal results wereobtained when a total dose of 100-150 mg (approx 5 mg/g body wt.) wasadministered in 0.3 ml of sterile, pyrogen-free saline. Qualtitativelysimilar results were obtained when sepsis was induced with 75 or 200 mg,with delayed or accelerated sickness, respectively. Two experiments wereperformed with 5 mice per group. NAC (Sigma, St. Louis. Mo.) was givenintraperitonealy at a dose of 0.27 mg/g of body weight at a single site.Antisense NF-_(κ)B p65 ODN (sequence 5′-AAACAGATCGTCCATGGTCA-3′) was 3′terminal phosphorothioate (PS)-modified (Kitajima, I., et al., Science,259:1523, 1993; and Winer, B. J. 1971. Statistical Principles InExperimental Design. McGraw-Hill New York, N.Y.), as this has been shownto confer stability in animals (Kitajirna, I., et al., J. Biol. Chem.267:25881, 1992). Extensive previous experience with this ODN, incomparision with NF-kB sense and irrelevant antisense controls has shownit to be highly specific, both in vivo and in vitro (Kitajima, I., etal., Science 258:1792, 1992). The p65 AS ODN preserves this specificityeven when used at doses twice the concentration used in this study.Antisense ODNs were administered ip at a dose of 40 mg/g of body weight,as it has been demonstrated that in previous in vivo studies (Kitajima,I., et al., J. Biol. Chem. 267:25881, 1992) this is an effective dose.AS-ODNs were administered i.p. in a volume of 0.3 ml sterile saline,while no treatment controls received only 0.3 ml of sterile saline. Thenumber of surviving mice in each treatment group was recorded beginningat 12 hrs and additional observations were performed at 24, 48, 72 and96 hours. The significance of differences in the survival rates betweengroups was evaluated by a Chi square test using all data points otherthan zero time. This provides an extremely conservative analysis inwhich a p value less than 0.05 is highly significant (Winer, B. J. 1971.Statistical Principles In Experimental Design. McGraw-Hill New York,N.Y.).

[0062] Control mice injected with LPS only, became ill within a fewhours. This was manifested as decreased spontaneous activity, followedby hunching and decreased oral intake. Two of ten mice died by 12 hoursand all of these mice ultimately died by 48 hours after a singleintraperitoneal injection of 5 mg/g body wt (100-150 mg) (solid bars).Mice which were pretreated with NAC prior to LPS challenge showedincreased but not significant protection from lethality (Total ChiSquare 8.31, p=0.08), in agreement with previous studies (Peristeris,P., et al., Cellular Immunology 140:390, 1992; Broner, C. W., et al.,Critical Care Medicine 16:848, 1988) (striped bars). Ultimate survivalrate was 40% with one death occuring prior to 12 hours. Mice pre-treatedwith antisense NF-_(κ)B ODNs had a significantly better outcome (TotalChi square 11.86 p=0.018) (dotted bars). Deaths were delayed beyond 12hours and the ultimate survival rate was 70%. Treatment with taxantisense or NF-_(κ)B sense control ODNs (Kitajima, I., et al., Science.258:1792, 1992; Kitajima, I., et al., J. Biol. Chem. 267:25881, 1992)gave results identical to the untreated control.

EXAMPLE 4 SERUM LEVELS OF IL-6 ARE DECREASED BY NF-_(κ)B ANTISENSE ODNsWHEN ADMINISTERED PRIOR TO SEPTIC SHOCK

[0063] The results of Example 3 suggest NF-_(κ)B antisense inhibitiondramatically reduces LPS induced lethality. In order to confirm thatthis is a specific consequence of NF-_(κ)B inhibition, the level ofserum IL-6 was measured. Previous studies have demonstrated that IL-6serves as an accurate indicator for NF-_(κ)B activation as this is aprimary transcriptional activator of this cytokine (Liberman, T., etal., Cell. Biol. 10:2327, 1990). In addition, IL-6 is an importantmediator of catastrophic immune responses such as inflammatorynephropathy (Horii, Y., et al., J. Immunol. 143:3949, 1989; Rugo, H. S.,et al., J. Clin. Invest. 89:1032, 1992), may modulate immunoreactivityof tumors (Tabibzadeh, S. S., et al., Am. J. Pathol. 135:1025, 1989),and has been strongly implicated in the pathogenesis of septic shock(Ulich, T. R., et al., J. Immunol 146:2316, 1991; Troutt, A. B. et al.,J. Cellular Physiology, 138:38, 1989).

[0064] Assay for IL-6 protein in serum. Sera from mice within eachtreatment group were obtained at 0, 1, 4, 8, and 20 hrs after LPSadministration. Serum IL-6 was determined by an enzyme-linkedimmunosorbent assay (ELISA) according to the method of Pruslin et al.(Pruslin, F. H., et al., J. Immunol. Method 137:27, 1991) using ratanti-mouse monoclonal antibodies to IL-6 (Pharmingen, San Diego,Calif.). The detection limit for this assay was 100 pg/ml. Statisticaldifferences between treatment groups were analyzed by the ANOVA test fortime points beyond zero. Statistical analysis was performed using theprogram Statview, on a Macintosh computer.

[0065] Serum IL-6 protein levels were measured by ELISA. Three mice wereused per treatment group and experiments were repeated twice.Inter-experiment variability was less than 20%. Data were pooled fromthese experiments and are displayed in FIG. 7 (No Rx, LPS given at timezero without specific inhibitor. NAC, pretreatment as described inmethods. NF-_(κ)B, pretreatment with ODN. No LPS, mice given sterilesaline only at corresponding times). IL-6 levels rapidly rose from thelower limit of detectability (100 pg/ml) at time zero to peak levels(5800 pg/ml) at 4 hrs after ip injection of LPS. Levels remained high(3900 pg/ml) up to 20 hrs after treatment. In contrast, serum levels ofIL-6 were almost two fold (51%) lower at 4 hrs after LPS challenge (2900pg/ml) in mice pretreated with antisense NF-_(κ)B ODNs. The serum IL-6levels at 4 hours in mice pretreated with NAC were not significantlydifferent from the LPS only control (74% or 4300 pg/ml) (FIG. 7).

[0066] ANOVA analysis of the 3 groups at 4, 8 and 20 hours, revealed areduction of IL-6 levels by NAC which was barely significant [p=0.03,F(1,24)=6.68], and a highly significant reduction by NF-_(κ)B antisenseODN [p=0.0001, F(1,24)=71.-61].

EXAMPLE 5 TISSUE SPECIFIC EFFECT OF NF-_(κ)B ANTISENSE ODNs ON LPSINDUCED IL-6 mRNA

[0067] Previous studies in mice have suggested nonuniform accumulationof oligonucleotides in different tissues when administeredintraperitoneally or intravenously (Iversen, P. et al., Anti-cancer DrugDesign 6:531, 1991). Previous studies suggested that oligonucleotideuptake by cells may be highly dependent on activation state (Kitajima,I., et al., J. Biol. Chem. 267:25881, 1992). Therefore, the effects ofNF-_(κ)B antisense on IL-6 in individual tissues in the presence orabsense of LPS activation were evaluated. IL-6 mRNA expression wasmonitored because previous studies have shown that its expression may bepleomorphically induced in a large number of tissues (Ulich, T. R., etal., J. Immunol 146:2316, 1991; Trouft, A. B. et al., J. CellularPhysiology, 138:38, 1989) and can therefore be used to assess NF-_(κ)Binhibition in variety of tissues.

[0068] Northern blot analysis. Kidney, liver, lung, spleen and salivaryglands from representative mice of each group were rapidly frozen inliquid nitrogen and crushed with a sterile pestle while frozen. mRNA wasextracted using the Fast Track method (Bradley, J. E., et al.,BioTechniques 6:114, 1988) (Invitrogen, San Diego, Calif.). The positivecontrol for murine gene expression (TNF-α, IL-6, MHC class I andGM-CSF), was mRNA obtained from an HTLV-I tax expressing mousefibrosarcoma cell line (Kitajima, I., et al., J. Biol. Chem. 267:25881,1992). 7 mg mRNA per lane was loaded and electrophoresis was performedon formaldehyde 1.2% agarose gels. Samples were blotted onto nylonmembrane and fixed by UV crosslinking. Murine cDNA probes of MHC class I& II and GM-CSF were labeled via the random primer method with[³²P]dATP. IL-6 TNF-α and actin probes cloned in pUC, pBluescript orpGem plasmids were labeled with [³²P]dATP by polymerase chain reactionusing universal primers complementary to regions flanking the linkers.Specific activities of probes were in excess of 1×10⁹ cpm/mg.

[0069] mRNAs were extracted from each tissue, 3 hrs after LPS injection.In FIG. 8, the lanes were as follows: B is an HTLV-I tax transformedcell line which expresses high levels of NF-_(κ)B and serves as apositive control. No is obtained from mock (saline treated) mice. LPS,obtained after no pretreatment. NAC, is from pretreated mice (8 mg×2).NF is from art animal pretreated with 40 mg/g NF-_(κ)B antisense ODN.DNA probes (IL-6, TNF-α, MHC class I, GM-CSF and actin) are indicated atthe left. Analysis of liver and kidney are shown in panel A and lung,salivary gland and spleen are shown in panel B. Lanes 1-9 representpretreatment for 3 hours with NAC or antisense NF-_(κ)B ODN. Lanes 10-18show the effects of 24 hrs pretreatment.

[0070] Pretreatment by antisense NF-_(κ)B ODNs caused a profounddecrease in LPS induced IL-6 mRNA in kidney (FIG. 8A, compare lanes 18to 16 and 9 to 7), with somewhat less effect in lung (FIG. 8B comparelanes 5 to 3), and lower effect in liver or other tissues. The uptakeand inhibitory effect of the oligonucleotides was time dependent aseffects were much greater when ODNs were administered 20 hrs rather than3 hrs prior to LPS (compare right and left hand portions of FIG. 8A).NAC pre-treatment inhibited IL-6 expression in a qualitatively similarmanner but was less effective in most tissues tested.

[0071] To further confirm the specificity of NF-_(κ)B inhibition intissues, mRNA of two additional genes were analyzed, TNF-α and MHC class1, both known to be NF-kB dependent and implicated in the pathogenesisof sepsis or catastrophic immune responses. Both antisense and NACdown-modulated expression of these genes though the effect was less thanthat for IL-6 (FIG. 8). FIGS. 8A and B show that IL-6 mRNA expressionwas highly induced by LPS in liver, kidney and spleen, moderatelyinduced in lung and weakly induced in salivary gland. IL-6 expressionwas not detected in brain and muscle.

[0072] The invention now being fully described, it will be apparent toone of ordinary skill in the art that various changes and modificationscan be made without department from the spirit or scope of theinvention.

1. An antisense oligonucleotide complementary to a region of a nuclearfactor-_(κ)B(NF-_(κ)B) subunit mRNA comprising a nucleic acid sequencehybridizing to the mRNA.
 2. The antisense oligonucleotide of claim 1,which comprises a nucleic acid sequence complementary to the translationinitiation nucleic acid sequence of the NF-_(κ)B subunit.
 3. Theantisense oligonucleotide of claim 1, which is from about 8 to about 40nucleic acid residues in length.
 4. The antisense oligonucleotide ofclaim 1, which is chemically modified.
 5. The antisense oligonucleotideof claim 4, which is chemically modified by substitution in anon-bridging oxygen atom of the antisense nucleic acid backbone with amoiety selected from the group consisting of methane phosphate, methylphosphate, and phosphorothioate.
 6. The antisense oligonucleotide ofclaim 5, wherein the substitution is at a terminal region selected fromthe group consisting of the 3′ region and the 5′ region.
 7. Theantisense oligonucleotide of claim 6, wherein the terminal region is the3′ terminal region.
 8. The antisense oligonucleotide of claim 1, whereinthe NF-_(κ)B subunit, to which the antisense nucleic acid is against, isselected from the group consisting of p65 and p50.
 9. The antisenseoligonucleotide of claim 1, wherein the antisense nucleic acid is DNA.10. The antisense oligonucleotide of claim 1, wherein the antisensenucleic acid is RNA.
 11. The antisense oligonucleotide of claim 1capable of hybridizing to a DNA sequence selected from the groupconsisting of: 3′ TAG CAG ACG GTA CCA CTT CTA 5′; 3′ CTT GTC AAG CAG GTACCG GC 5′; and 3′ TTT GTC TAG CAG GTA CCA GT 5′.


12. The antisense oligonucleotide of claim 11, which is selected fromthe group consisting of: 5′ ATC GTC TGC CAT GGT GAA GAT 3′; 5′ GAA CAGTTC GTC CAT GGC CG 3′; 5′ AAA CAG ATC GTC CAT GGT CA 3′; and 5′ TC GTCTGC CAT GGT GAA GAT 3′.


13. A recombinant DNA sequence which, upon translation, produces anantisense oligoribonucleotide against a NF-_(κ)B subunit mRNA, whereinthe antisense oligoribonucleotide comprises a nucleic acid sequencehybridizing to the mRNA.
 14. A method of suppressing NF-_(κ)B dependentprocesses in an individual which comprises administering atherapeutically effective amount of the antisense oligonucleotide ofclaim
 1. 15. The method of claim 14, wherein the antisenseoligonucleotide is complementary to the translation initiation nucleicacid sequence of the NF-_(κ)B subunit.
 16. The method of claim 14,wherein the antisense oligonucleotide is from about 8 to about 40nucleic acid residues in length.
 17. The method of claim 14, wherein theantisense oligonucleotide is chemically modified.
 18. The method ofclaim 17, wherein the antisense oligonucleotide is chemically modifiedby substitution in a non-bridging oxygen atom of the antisenseoligonucleotide backbone with a moiety selected from the groupconsisting of methane phosphate, methyl phosphate, and phosphorothioate.19. The method of claim 18, wherein the antisense oligonucleotide issubstituted at a terminal region selected from the group consisting ofthe 3′ region and the 5′ region.
 20. The method of claim 19, wherein theantisense oligonucleotide terminal region is the 3′ terminal region. 21.The method of claim 14, wherein the NF-_(κ)B subunit, to which theantisense oligonucleotide is against, is selected from the groupconsisting of p65 and p50.
 22. The method of claim 14, wherein theantisense oligonucleotide is DNA.
 23. The method of claim 14, whereinthe antisense oligonucleotide is RNA.
 24. The method of claim 14,wherein the antisense oligonucleotide is capable of hybridizing to a DNAsequence selected from the group consisting of: 3′ TAG CAG ACG GTA CCACTT CTA 5′; 3′ CTT GTC AAG CAG GTA CCG GC 5′; and 3′ TTT GTC TAG CAG GTACCA GT 5′.


25. The method of claim 24, wherein the antisense oligonucleotide isselected from the group consisting of: 5′ATC GTC TGC CAT GGT GAA GAT 3′;5′GAA CAG TTC GTC CAT GGC CG 3′; 5′AAA CAG ATC GTC CAT GGT CA 3′; and5′TC GTC TGC CAT GGT GAA GAT 3′.


26. The method of claim 14, wherein the antisense oligonucleotide isadministered in combination with a glutathione precursor.
 27. The methodof claim 26, wherein the antisense oligonucleotide and glutathioneprecursor are administered substantially contemporaneously.
 28. Themethod of claim 26, wherein the glutathione precursor is anacylcysteine.
 29. The method of claim 28, wherein the acylcysteine isN-acetylcysteine.
 30. A pharmaceutical combination comprising theantisense oligonucleotide of claim 1, in combination with a glutathioneprecursor.
 31. The method of claim 14, wherein the NF-_(κ)B dependentprocess is septic shock.
 32. The method of claim 14, wherein theNF-_(κ)B dependent process is tissue specific.
 33. The method of claim32, wherein the tissue is selected from the group consisting of kidney,liver, and spleen.
 34. A method of monitoring the effectiveness ofsuppressing NF-_(κ)B dependent processes in the tissue of an individualafter administering a therapeutically effective amount of the antisenseoligonucleotide of claim 1, comprising detecting the level of cytokineproduction in a tissue before and after the antisense therapy.
 35. Themethod of claim 34, wherein the cytokine is IL-6.